Various publications, including patents, published applications, technical articles, scholarly articles, and gene or protein accession numbers are cited throughout the specification. Each of these materials is incorporated by reference herein, in its entirety and for all purposes.
CD38 is a 46 kDa type II transmembrane glycoprotein. It has a short N-terminal cytoplasmic tail of 20 amino acids, a single transmembrane helix and a long extracellular domain of 256 amino acids. It is expressed on the surface of many immune cells including CD4 and CD8 positive T cells, B cells, NK cells, monocytes, plasma cells and on a significant proportion of normal bone marrow precursor cells. In some instances, the expression of CD38 in lymphocytes may be dependent on the differentiation and activation state of the cell, for example, resting T and B cells may be negative while immature and activated lymphocytes may be predominantly positive for CD38 expression. CD38 mRNA expression has been detected in non-hemopoeitic organs such as the pancreas, brain, spleen and liver (Koguma, T. (1994) Biochim. Biophys. Acta 1223:160).
CD38 is a multifunctional ectoenzyme that is involved in transmembrane signaling and cell adhesion. It is also known as cyclic ADP ribose hydrolase because it can transform NAD+ and NADP+ into cADPR, ADPR and NAADP, depending on extracellular pH. These products induce Ca2+-mobilization inside the cell, which can lead to tyrosine phosphorylation and activation of the cell. CD38 is also a receptor that can interact with a ligand, CD31. Activation of receptor via CD31 leads to intracellular events including Ca2+ mobilization, cell activation, proliferation, differentiation and migration.
CD38 is expressed at high levels on multiple myeloma cells, in most cases of T- and B-lineage acute lymphoblastic leukemias, some acute myelocyticleukemias, follicular center cell lymphomas and T lymphoblastic lymphomas. CD38 is also expressed on B-lineage chronic lymphoblastic leukemia (B-CLL) cells. In some cases, B-CLL patients presenting with a CD38+ clone are characterized by an unfavorable clinical course with a more advanced stage of disease, poor responsiveness to chemotherapy and shorter survival time. The use of antibodies to CD38 has been proposed for the treatment of CD38-expressing cancers and hematological malignancies. It may therefore be advantageous to provide alternative antibodies to CD38 which have desirable manufacturing, stability and immunogenic properties.
Numerous peptide and polypeptide ligands have been described to function by interacting with a receptor on a cell surface, and thereby stimulating, inhibiting, or otherwise modulating a biological response, usually involving signal transduction pathways inside the cell that bears the said receptor. Examples of such ligands include peptide and polypeptide hormones, cytokines, chemokines, growth factors, and apoptosis-inducing factors.
Due to the biological activities of such ligands, many have potential uses as therapeutics. Several peptide or polypeptide ligands have been approved by regulatory agencies as therapeutic products including, for example, human growth hormone, insulin, interferon (IFN)-alpha2b, IFN-alpha2a, IFNβ, erythropoietin, G-CSF and GM-CSF.
While these and other ligands have demonstrated potential in therapeutic applications, they may also exhibit toxicity when administered to human patients. One reason for toxicity is that most of these ligands trigger receptors on a variety of cells, including cells other than those that mediate the desired therapeutic effect. A consequence of such “off target” activity of ligands is that many ligands are currently not suitable for use as therapeutic agents because the ligands cannot be administered at sufficiently high dosages to produce maximal or optimal therapeutic effects on the target cells which mediate the therapeutic effect.
For example it has been known since the mid-1980's that interferons, in particular IFN-alpha, are able to increase apoptosis and decrease proliferation of certain cancer cells. IFN-alpha has been approved by the FDA for the treatment of several cancers including melanoma, renal cell carcinoma, B cell lymphoma, multiple myeloma, chronic myelogenous leukemia (CML) and hairy cell leukemia. A direct effect of IFN-alpha on the tumor cells is mediated by the IFN-alpha binding directly to the type I IFN receptor on those cells and stimulating apoptosis, terminal differentiation or reduced proliferation. A further indirect effect of IFN-alpha on non-cancer cells is to stimulate the immune system, which may produce an additional anti-cancer effect by causing the immune system to reject the tumor.
These biological activities are mediated by type I interferon receptors on the surface of the cancer cells which, when stimulated, initiate various signal transduction pathways leading to reduced proliferation and/or the induction of terminal differentiation or apoptosis. The type I interferon receptor is, however, also present on most non-cancerous cells. Activation of this receptor on non-cancerous cells by IFN-alpha causes the expression of numerous pro-inflammatory cytokines and chemokines, leading to toxicity and untoward effects. Such toxicity may cause severe flu-like symptoms, which prevents the dosing of IFN-alpha to a subject at levels that exert the maximum anti-proliferative and pro-apoptotic activity on the cancer cells.
When IFN-alpha2b is used to treat multiple myeloma, its utility resides, at least in part, in its binding to type I interferon receptors on the myeloma cells, which in turn triggers apoptosis and/or reduced proliferation and hence limits disease progression. Unfortunately, however, this IFN also binds healthy cells within the body, triggering a variety of other cellular responses, some of which are harmful.
A publication by Ozzello (Breast Cancer Research and Treatment 25:265-76, 1993) describes chemically conjugating human IFN-alpha to a tumor-targeting antibody, thereby localizing the direct inhibitory activity of IFN-alpha to the tumor as a way of reducing tumor growth rates, and demonstrated that such conjugates have anti-tumor activity in a xenograft model of a human cancer. The mechanism of the observed anti-cancer activity was attributed to a direct effect of IFN-alpha on the cancer cells, since the human IFN-alpha used in the experiments did not interact appreciably with the murine type I IFN receptor, which could have led to an indirect anti-cancer effect. Because of this lack of binding of the human IFN-alpha to the murine cells, the toxicity of the antibody-IFN-alpha conjugate relative to free INF-alpha was not assessed.
Antibodies and IFN-alpha may also be connected together in the form of a fusion protein. For example, WO 01/97844 describes a direct fusion of human IFN-alpha to the C-terminus of the heavy chain of an IgG specific for the tumor antigen CD20.
In general, IFN may be targeted to cancer cells. While this approach may result in an increase in activity of the IFN against cancer cells, it does not completely address the issue of undesired activity of the IFN on healthy cells. Fusing IFN-alpha to the C-terminus of the heavy chain of an IgG may prolong the half-life of the IFN alpha leading to undesirable adverse events. Accordingly, there exists a need to decrease off-target activity of ligand-based drugs, while retaining the “on-target” therapeutic effect of such ligands.